DNA sequences coding for mycoplasma hyopneumoniae surface antigens, corresponding proteins and use in vaccines and diagnostic procedures

ABSTRACT

Mycoplasma hyopneumoniae P65 surface antigens prepared by recombinant DNA or synthetic methods, protein antigens encoded by P65 gene, an expression vector and transformed host containing the antigens, a vaccine based on such antigens, methods of treating swine, etc. to prevent enzootic pneumonia using that vaccine and diagnostic tests to detect the presence of Mycoplasma hyopneumoniae.

This is a continuation of application Ser. No. 08/373,957, Jan. 17, 1995 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to Mycoplasma hyopneumoniae P65 surface antigen, a lipoprotein capable of eliciting an antibody or other immune response which selectively recognizes an epitope(s) of P65, to such antigens prepared by recombinant DNA methods, to swine mycoplasma vaccine based on such antigens, to methods of treating swine to prevent enzootic pneumonia using that vaccine, and to diagnostic tests for detecting the presence of swine mycoplasma infections in swine herds.

2. Brief Description of the Prior Art

Enzootic pneumonia in swine is caused by Mycoplasma hyopneumoniae. The disease is transmitted from pig to pig through the nasal passages by airborne organisms expelled from the lungs of infected pigs. The primary infection by Mycoplasma hyorneumoniae may be followed by secondary infection by other mycoplasma species (Mycoplasma hyorhinis and Mycoplasma flocculare) as well as bacterial pathogens. The disease rarely causes death but causes decreased growth and weight gain.

Mycoplasma hyopneumoniae is a small, prokaroytic microbe smaller and simpler in structure than bacteria, but more complex than viruses. Unlike viruses, they are capable of a free living existence, through they are often found in association with eukaroytic cells as they have absolute requirements for exogenous sterols and fatty acids which generally necessitates growth in serum-containing media. Mycoplasma hyopneumoniae is bounded by a cell membrane but not by a cell wall. They have an extremely small genome, approximately 750,000 base pairs in length.

Due to the serious economic consequences of pig pneumonia, vaccines against Mycoplasma hyopneumoniae and diagnostic testing methods which will indicate the presence of an infection have been sought. Vaccines containing preparations of mycoplasma organisms grown in broth medium have been marketed. However, the requirement for serum in growth medium adds great expense to the process and introduces possible concerns regarding adverse reactions induced by serum components present in the immunizing material. Cost effective production of the appropriate recombinant mycoplasma protein in the absence of serum offers an improvement in terms of economy and vaccine safety. Such a recombinant protein also offers a specific target for recognition of antibodies to the organisms, which may detect infection with this agent. Current serologic assays for detection are confounded by non-specific reactions due to the antigenic cross-reactivity of multiple common swine mycoplasmas.

In earlier work, certain lipoproteins present on the cellular membrane of Mycoplasma hyopneumoniae were identified as mediating or inducing immunological reactions (Kim, M. F., M. B. Heidari, S. J. Stull, M. A. McIntosh, and K. S. Wise. 1990. Infect. Immun. 58:2637-2643; Wise, K. S., and M. F. Kim. 1987 J. Bacteriol. 169:5546-5555). From those lipoproteins, P65 was shown to be capable of eliciting an antibody which selectively recognized an epitope(s) of P65 without unwanted cross-reactions with antibodies to other mycoplasmas, especially Mycoplasma flocculare. P65 was shown to be integrally associated with the cell membrane and covalently linked to a lipid moiety, with the majority of the protein, including the C-terminus, exposed to the exterior of the cell, consistent with the predicted orientation of surface lipoproteins which are anchored by N-terminal lipid moieties.

While P65 was an attractive candidate for developing a recombinant protein because of its ability to elicit an antibody which is selective for Mycoplasma hyooneumoniae, attempts to determine the amino acid sequences failed because of N-terminal blocking by the lipid and isolation of internal fragments has been problematic. Functional analyses have been similarly unrewarding because of the attached lipid which renders P65 extremely hydrophobic making detergents an absolute requirement for solubilization which essentially precludes tests on viable cells or those tests involving aqueous buffer systems.

Determining and utilizing the DNA sequence coding for P65 has also been very difficult, despite the small size of the mycoplasma chromosome, because of a lack of vehicles for mobilizing and transferring DNA into these organisms (Kim et al, 1990, supra) and because of inherent problems in the expression of mycoplasma genes in more versatile, heterologous hosts such as E. coli. Mycoplasma hyopneumoniae uses an alternative genetic code (in which the UGA translation termination codon is used to encode tryptophan) and expression of mycoplasma genes in E. coli is complicated by truncation of products at the UGA codons. In addition, expression of partial products occurs through promiscuous translation from multiple, unpredictable sites in structural genes, a phenomenon that is probably common among most mycoplasmas (Notarnicola, S. N., M. A. McIntosh, and K. S. Wise. 1990. J. Bacteriol. 172:2986-2995).

SUMMARY OF THE INVENTION

In view of the above, the present invention relates to a DNA sequence shown in FIG. 1. The invention also relates to a biologically active fragment of said DNA sequence which encodes at least one protein which is capable of eliciting an antibody or other immune response which recognizes an epitope(s) of P65 and to a biologically active mutant of said DNA sequence which encodes at least one protein which is capable of eliciting an antibody or other immune response which recognizes an epitope(s) of P65. The subject DNA sequence may encode a protein which is the entire antigen, or a fragment or derivative of the antigen or a fusion product of the antigen or fragment and another protein, provided that the protein which is produced from such DNA sequence is capable of eliciting an antibody or other immune response which recognizes an epitope(s)of P65.

The present invention also concerns an essentially pure protein having a sequence coded by any of the DNA sequences described above, including the sequence shown in FIG. 2; an expression vector containing a promoter, a non-coding sequence and any of the coding DNA sequences described above and a host transformed with said expression vector; a vaccine containing a protein having a sequence coded by any of the DNA sequences described above; a diagnostic procedure based on those proteins or antibodies raised against them for detecting the presence of Mycoplasma hyopneumoniae infections in swine herds, and a diagnostic procedure utilizing any portion of said DNA sequence shown in FIG. 1 or an RNA equivalent thereof in RNA form to detect the presence of the gene or organism in swine-derived material by methods of synthesis, amplification or hybridization of nucleic acid sequences.

The invention summarized above comprises the DNA and protein sequences, etc. hereinafter described, the scope of the invention being indicated by the subjoined claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1G depicts the DNA sequence encoding the entire 627 amino acids of the structural gene for the surface lipoprotein P65 from Mycoplasma hyopneumoniae and includes 312 base pairs upstream and 479 base pairs downstream of the coding sequence. The DNA sequence in the coding sequence is divided into three base codons which align with the proper reading frame of the gene. SEQ ID NO:1 The amino acid sequence (lower line) is the translation of the DNA codon directly above with all UGA codons assigned as tryptophan.

FIG. 2A-2D depicts the amino acid sequence of the intact surface lipoprotein P65. SEQ ID NO:2

FIG. 3 is a diagram of plasmid pZJ25.1A. The hatched region represents a DNA sequence responsible for the expression of a polypeptide (P19) carrying epitopes related to P65.

FIG. 4 is a diagram of plasmid pZJ35.1A. The region shown as an open box represents the 2672-bp fragment for which the entire nucleotide sequence is given in FIG. 1 (SEQ ID NO:1) and contains the complete P65 structural gene.

FIG. 5 is a schematic showing the structure and features of Mycoplasma hyopneumoniae P65 lipoprotein gene.

FIG. 6 is a diagram of expression of the vector pMAL-c2. The P65 structural gene was cloned into this vector as a BamHI-HindIII fragment ligated to the corresponding sites in the vector polylinker region.

FIG. 7 is a schematic showing the purification of rP65.

FIG. 8 shows the recognition of recombinant forms of P65 by the Mab defining this protein, and the selective recognition of the recombinant forms by swine infected with Mycoplasma hyopneumoniae. The three panels are Western blots of identical samples, immunostained with the Mab defining P65 (left panel), serum from swine after experimental inoculation with Mycoplasma hyopneumoniae (central panel), or serum from the same swine prior to inoculation (right panel). Antibodies and sera are described in Wise and Kim, 1990, supra; and Kim et al, 1990, supra. Each panel represents three preparations: affinity purified fusion protein FP65 (left lane of each panel), the same preparation treated with factor Xa to generate the rP65 fragment (arrows) (middle lane of each panel), or Triton X-114 phase membrane proteins of Mycoplasma hyopneumoniae (right lane of each panel). Minor bands in the left panel are breakdown products of the predominant FP65 or rP65 proteins, detected by sensitive Mab staining.

FIG. 9 shows the solubility properties of authentic and recombinant P65 after TX-114 fractionation by Western blot analysis.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with the drawings and the following examples, serve to explain the principles of the invention. All references discussed in this specification are hereby incorporated in their entirety by reference. The three letter designations for amino acids used in this application are as follows:

    ______________________________________                                                       THREE-LETTER                                                     AMINO ACID    ABBREVIATION                                                     ______________________________________                                         Alanine       Ala                                                              Arginine      Arg                                                              Asparagine    Asn                                                              Aspartic acid Asp                                                              Cysteine      Cys                                                              Glutamine     Gln                                                              Glutamic acid Glu                                                              Glycine       Gly                                                              Histidine     His                                                              Isoleucine    Ile                                                              Leucine       Leu                                                              Lysine        Lys                                                              Methionine    Met                                                              Phenylalanine Phe                                                              Proline       Pro                                                              Serine        Ser                                                              Threonine     Thr                                                              Tryptophan    Trp                                                              Tyrosine      Tyr                                                              Valine        Val                                                              ______________________________________                                    

As noted above, the present invention relates to a recombinant DNA sequence between base pairs 313 and 2193 as shown in FIG. 1, (SEQ ID NO:1) the complete 2672 sequence illustrating the entire structural gene for Mycoplasma hyopneumoniae P65 including its hydrophobic lipid-modified N-terminal end. More generally, the invention concerns any DNA sequence coding for a protein which is capable of eliciting an antibody or other immune response (e.g., T-cell response of the immune system) which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2), including less than the full DNA sequence and mutants thereof. Hence the DNA sequence may encode a protein which is the entire antigen between base pairs 313 and 2193, or a fragment or derivative of the antigen or a fusion product of the antigen or fragment and another protein, provided that the protein which is produced from such DNA sequence is capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2.

As a result, the term "DNA sequence coding for a protein which is capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2" (SEQ ID NO:2) encompasses DNA sequences which encode for and/or express in appropriate transformed cells, proteins which may be the full length antigen, antigen fragment, antigen derivative or a fusion product of such antigen, antigen fragment or antigen derivative with another protein. Included antigen derivatives include those where UGA codons have been converted into codons recognized by the host as non stop codons, most commonly UGG.

Proteins included within the present invention have an amino acid sequence depicted in FIG. 2 (SEQ ID NO:2). Other included proteins consist of a fragment of said sequence capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2) and a mutuant of said sequence capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2).

The appropriate DNA sequence may be inserted into any of a wide variety of expression vectors by a variety of procedures, in general, through an appropriate restriction endonuclease site. Such procedures and others are deemed to be known by those skilled in the art. Suitable vectors include chromosomal, nonchromosomal and synthetic DNA sequences; e.g., derivatives of SV40; bacterial plasmids; phage DNAs; yeast plasmids; vectors derived from combinations of plasmids and phage DNAs, viral DNA such as baculovirus, vaccinia, adenovirus, fowl pox virus, pseudorabies, etc. The appropriate DNA sequence must be operatively linked in the vector to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned LTR or SV40 promoter, the E. coli lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic and eukaryotic cells or their viruses. The expression vector also includes a non-coding sequence for a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. Examples of host organisms and cells include bacterial strains (e.g., E. coli, Pseudomonas, Bacillus, Salmonella, etc.), fungi (e.g., yeasts and other fungi), animal or plant hosts (e.g., mouse, swine or animal and human tissue cells). The selection of the host is deemed to be within the scope of those skilled in the art.

It is also understood that the appropriate DNA sequence present in the vector when introduced into a host may express part or only a portion of the protein which is encoded within the noted terminology, it being sufficient that the expressed protein be capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2). For example, in employing E. coli as a host organism, the UGA codon is a stop codon so that the expressed protein may only be a fragment of the antigen encoded into the vector and for this reason it is generally preferred that all of the UGA codons in the appropriate DNA sequence be converted into non stop codons. Another way around the problem in a host that recognizes UGA as a stop codon is to include an additional DNA sequence which encode a t-RNA which translates the UGA codon within a protein coding sequence as tryptophan in the transformed organism.

The protein expressed by the host transformed by the vector containing the appropriate DNA sequence containing one or more proteins which are capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2) may be harvested by methods which will occur to those skilled in the art and used in a vaccine for protection of a non-human animal, such as a bovine, swine, etc., against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae. Said one or more proteins which are capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2) are used in an amount effective to provide protection against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae and may be used in combination with a suitable physiologically acceptable carrier.

The term "protecting" or "protection" when used with respect to the vaccine for mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae described herein means that the vaccine prevents mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae or reduces the severity of the disease.

The carrier which is employed in conjunction with the protein antigen may be any one of a wide variety of carriers. As representative examples of suitable carriers, there may be mentioned mineral oil, synthetic polymers, etc. Carriers for vaccines are well known in the art and the selection of a suitable carrier is deemed to be within the scope of those skilled in the art. The selection of a suitable carrier is also dependent upon the manner in which the vaccine is to be administered.

The present invention provides a method of immunizing a susceptible non-human animal, e.g., swine, bovine, etc., against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae with the vaccine described above. For purposes of this invention, the vaccine is administered in an effective amount. The vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal or intravenous injection. Alternatively, the vaccine may be administered intranasally or orally. It is also to be understood that the vaccine may include active components or adjuvants in addition to the antigen(s) or fragments hereinabove described.

The host expressing the antigen may itself be used to deliver antigen to non-human animals, by introducing killed or viable host cells that are capable of propagating in the animal. Direct incorporation of P65 DNA sequences into host cells may also be used to introduce the sequences into animal cells for expression of antigen in vivo.

The present invention also provides a method for testing a non-human animal, e.g., swine, bovine, etc., to determine whether the animal has been vaccinated with the vaccine of the present invention, other vaccines containing P65 protein, or is infected with naturally-occurring Mycoplasma hyopneumoniae. This method comprises obtaining from the animal a sample of suitable body fluid or tissue, detecting in the sample a presence of antibodies or other immune responses to Mycoplasma hyopneumoniae by reaction with a protein having an amino acid sequence depicted in FIG. 2 (SEQ ID NO:2), a fragment of said sequence capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2) or a mutant of said sequence capable of eliciting an antibody or other immune response which recognizes an epitope(s) of the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2), the absence of such antibodies or other immune responses indicating that the animal has been neither vaccinated nor infected.

Alternatively, the fluid sample may be tested for the presence of a gene for Mycoplasma hyopneumoniae P65 by reaction with a recombinant or synthetic DNA sequence contained within the sequence shown in FIG. 1 (SEQ ID NO:1) or any RNA sequence, in RNA form, equivalent to said DNA sequence contained within the sequence shown in FIG. 1 (SEQ ID NO:1), the absence of said gene indicating that the animal has been neither vaccinated nor infected. Said test involving methods of synthesis, amplification or hybridization of nucleic acid sequences known within the scope of those skilled in the art.

EXAMPLES Identification, Characterization and Modification of the Mycoplasma Hyopneumoniae P65 Structural Gene

To isolate, clone and sequence the structural gene for the P65 lipoprotein from M. hyopneumoniae, a polyclonal antiserum to P65 was used to screen a recombinant phage library after infection of E. coli cells for the expression of any peptide epitope related to the M. hyopneumoniae P65 lipoprotein. The entire corresponding P65 gene was then isolated from overlapping chromosomal DNA fragments and characterized in detail, including its nucleotide sequence and its expression capability in E. coli. To manipulate the nucleotide sequence of this gene so that protein products related to P65 could be generated using universal expression systems in a heterologous host like E. coli, the DNA sequence was modified to standard codon usage, providing a foundation for overproduction and purification schemes described in the following section.

A recombinant phage from a M. hyopneumoniae genomic library was identified with antibodies to P65 and shown to produce a 19-kDa product (P19) that contained epitopes related to the authentic P65 lipoprotein (Kim et al., 1990, supra). DNA fragments from this phage were subcloned into the plasmid vector, pGEM3Z (Promega, Madison, Wis.) and one clone, pZJ25.1A (FIG. 3), which contained a 5.4-kb EcoRI fragment, was shown to express the P19 product. The sequence responsible for the expression of this 19-kDa C-terminal region of P65 was localized to an 800-bp region at one end of this fragment (shown in FIG. 3), but the remainder of the P65 gene was not contained in these sequences. An overlapping 5.0-kb HindIII fragment was isolated as the recombinant plasmid pZJ35.1A (FIG. 4) and shown to contain the entire P65 structural gene. This plasmid also expressed the P19 product and a P40 polypeptide, both containing epitopes related to P65 , but it did not express the full-length protein.

Nucleotide sequence was determined for a 2672-bp region of pZJ35.1A (the region noted in FIG. 4 and the sequence detailed in FIG. 1) (SEQ ID NO:1). The sequence encoded a 627-residue polypeptide (from nt313 to nt2193) which contained three internal, in-frame UGA codons (beginning at nt634, nt865, and nt1237) that designate tryptophan incorporation in mycoplasma proteins. These codons would prevent expression of the full-length P65 protein in E. coli. Downstream of these codons, positions for the internal initiation of translation leading to the synthesis of the P40 and P19 products were defined. The P65 coding region is shown schematically in FIG. 5. The 627-residue protein has a calculated molecular weight of 71,026, with a 29-amino acid hydrophobic signal sequence responsible for translocating the protein to the mycoplasma cell surface. The signal domain is bounded on its downstream end by the amino acid sequence SAGC which represents a typical lipoprotein processing site, leaving the C-residue modified (by fatty acid acylation) as the N-terminus of the mature protein. The calculated molecular weight of the mature protein, without modification, is 67,817.

In order to take advantage of the high-level E. coli expression systems to produce the full-length P65 product, the three in-frame UGA codons had to be changed to the universal tryptophan codon, UGG. The 1.3-kb EcoRI fragment containing all three UGA codons was removed from pZJ35.1A and cloned into the modified genome of the filamentous phage M13mp18 for oligonucleotide-directed mutagenesis. Oligonucleotides incorporating the UGA to UGG codon changes were generated for each of the three P65 gene regions, and standard reaction protocols were used to incorporate these changes into the P65 coding sequence. The mutagenized 1.3-kb EcoRI fragment was then removed from M13 and religated in place of the original fragment in pZJ35.1A to produce the mutated derivative pZJ35.1AM. This recombinant plasmid was shown to produce a P65 polypeptide in E. coli that contained the entire amino acid sequence of the mature mycoplasma protein and could be detected by polyclonal and monoclonal antibody (MAb) to P65.

Cloning and Overproduction of the Soluble Portion of P65 in an Expression Vector to Generate Two Forms of Recombinant P65.

To demonstrate that the full length P65 protein sequence could be expressed in a standard bacterial host with a conventional codon usage, and to show the feasibility of producing a soluble form of recombinant P65 that was abundant, easily purified and not denatured, a portion of the mutated P65 gene sequence was introduced into the expression vector pMAL-c2 (New England Biolabs, Beverly, Mass.)(NEB) shown in FIG. 6.

The overall strategy created a gene fusion between part of the P65 coding sequence and the vector-borne malE gene (encoding maltose binding protein, or MBP, and under control of the inducible tac promoter), to create a fusion protein (FP65 ) which accumulated in the cytoplasm of the E. coli host as a soluble protein. Affinity chromatography using immobilized amylose then rendered a purified FP65 , which was assessed directly, or was cleaved with the specific protease, Factor Xa (at a site encoded at the 3' end of the malE gene in the original pMAL-c2), to liberate the P65 -containing portion of the fusion protein, from the MBP-containing portion (refer to FIG. 7).

To ensure a soluble product, yet to include all external, hydrophilic sequences of the authentic mycoplasmal P65 protein, a selected region of the P65 gene was used for this fusion in which the signal peptide sequence and lipid acylation site were eliminated. Specifically, the mutated sequence encoded in the construct pZJ35.1AM (FIG. 4) was subcloned, using the HindIII site 3' of the P65 coding region, but a 5' site created by polymerase chain reaction to include the UGU(Cys) codon at position 400, and a BamHI site 5' to that codon, designed to create an in-frame fusion by ligation with a BamHI site in the polylinker region of pMAL-c2. The polymerase chain reaction primer used to create the P65 gene fusion junction was: 5'-GGA TCC TCA GCT GGT TGT TTG C-3'.

Demonstration of the Appropriate Recombinant Products, Recognition of Products by Antibodies of Swine Infected with Mycoplasma Hyopneumoniae and Solubility Properties

Demonstration of the appropriate recombinant protein products is indicated in FIG. 8, showing the results of SDS-PAGE and Western blot analysis of the recombinant proteins (using methods described in Wise and Kim, 1987, supra; and Kim et al, 1990, supra). Induction of the recombinant pMAL-c2 construct in the cell line E. coli DH5 alpha (Life Technologies, Inc., Gaithersburg Md.) resulted in specific expression of a pronounced high molecular weight product observed in Coomasie blue stained SDS gels. This protein was purified by amylose column chromatography, by methods provided by the manufacturer of pMAL-c2 (NEB), and identified as the predicted FP65 by staining with the Mab to authentic P65 (FIG. 8, left panel). This protein was also identified with Mab to MBP (Chemicon International, Inc., Temecula, Calif.)(data not shown). In addition, the protein was cleaved with Factor Xa to generate the predicted product, rP65 , shown by the arrows in the middle lanes in the left and central panels in FIG. 8. (Staining of these factor Xa digests with Mab to MBP also showed the predicted MBP portion of the fusion protein, at approximately 43 kDa; data not shown). The liberated rP65 product was slightly larger than the authentic mycoplasmal P65 , as predicted from additional residues encoded by vector sequences that are absent from the processed form of the mature protein in mycoplasma. This is shown by staining the rP65 product and detergent phase proteins from the mycoplasma (hpn TX) with Mab to P65 (compare the middle and right lanes of the left panel in FIG. 8). In summary, the recombinant forms of P65 were authenticated by selective amylose binding and purification of the fusion protein FP65 , identification by MAbs recognizing P65 epitopes and MBP epitopes on the FP65 product, and selective cleavage of FP65 with factor Xa to liberate the expected rP65 product, also identified with the defining Mab to P65.

Recognition of the FP65 product and the factor Xa-liberated rP65 product by antibodies of swine infected with M. hyopneumoniae is demonstrated in FIG. 8, in the central and right panels, which are Western blots of the same samples represented in the left panel. Using swine serum from animals either prior to (prechallenge) or after (post challenge) experimental infection and disease caused by M. hyopneumoniae (described in Kim et al, 1990, supra), both the FP65 and the liberated rP65 protein were selectively recognized. (compare middle and right panels). Despite the use of the Western blot technique, which significantly denatures the proteins, the recombinant forms of P65 displayed epitopes that were selectively recognized by infected swine. The undenatured form of purified FP65 and rP65 show the same selective recognition by infected swine, in a standard enzyme-linked immunosorbent (ELISA) assay (data not shown).

The solubility properties of both FP65 and rP65 are shown in comparison to the authentic, lipid modified mycoplasma version, using detergent fractionation (Wise and Kim, 1987, supra and Kim et al 1990, supra). FIG. 9 shows that the FP65 and the liberated rP65 partition to the aqueous phase (Aq), whereas the native hydrophobic P65 migrates to the detergent phase (Tx). This insures that the recombinant products are soluble and easily manipulated in standard aqueous buffers.

In view of the above, it will be seen that the several objectives of the invention are achieved and other advantageous results attained. As various changes could be made in the above DNA molecules, proteins, etc. without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 2                                                   (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2672 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: genomic DNA                                                (A) DESCRIPTION: region of 5.8 kb HindIII                                      fragment from genomic library                                                  (iii) HYPOTHETICAL: no                                                         (iv) ANTI-SENSE: no                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mycoplasma hyopneumoniae                                         (B) STRAIN: J                                                                  (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE:                                                               (G) CELL TYPE: unicellular bacterium                                           (H) CELL LINE:                                                                 (I) ORGANELLE:                                                                 (vii) IMMEDIATE SOURCE:                                                        (A) LIBRARY: Genomic in γ Charon 4A, γ GEM12                       (B) CLONE: γMhpJ25, γMhpJ35, γMhpJG35,                       pZJ25, pZJ25.1, pZ125.14,                                                      pZJG35.1, pZJG35.12, pZJG35.13,                                                pZJG35.14                                                                      (viii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: single chromosome                                      (B) MAP POSITION: unknown                                                      (C) UNITS: unknown                                                             (ix) FEATURE:                                                                  (A) NAME/KEY: sequence encodes entire 627                                      amino acids of the structural                                                  gene for the surface lipoprotein                                               P65 and includes 312 bp upstream                                               and 479 bp downstream of coding                                                sequence                                                                       (B) LOCATION: coding sequence for P65 spans                                    1881 bp of described sequence                                                  (beginsatnt313andincludes                                                      all sequence through nt 2193)                                                  (C) IDENTIFICATION METHOD: by similarity to pattern of open                    reading frame; by experiment                                                   identifying protein products of                                                sequence with immune serum to                                                  P65                                                                            (D) OTHER INFORMATION: immunogenic surface lipoprotein                         of no known function; C- terminus                                              exposed on external surface of                                                 cell                                                                           (x) PUBLICATION INFORMATION:                                                   (A) AUTHORS: Mary F. Kim, Manijeh B. Heidari,                                  Susan J. Stull, Mark A.                                                        McIntosh, and Kim S. Wise                                                      (B) TITLE: Identification and Mapping of an                                    Immunogenic Region of Mycoplasma                                               hyopneumoniae p65 Surface                                                      Lipoprotein Expressed in                                                       Escherichia coli from a Cloned                                                 Genomic Fragment                                                               (C) JOURNAL: Infection and Immunity                                            (D) VOLUME: 58                                                                 (E) ISSUE: 8                                                                   (F) PAGES: 2637- 2643                                                          (G) DATE: August 1990                                                          (H) DOCUMENT NUMBER:                                                           (I) FILING DATE:                                                               (J) PUBLICATION DATE:                                                          (K) RELEVANT RESIDUES IN SEQ ID NO: From 1 to 2672                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        AAGCTTGAGAAAATTACAAAAAAATTCATTTTCCAAAAACGCTTTTAGCT50                           TTTATTAAAGGGCCCAAAGTTTTTAATGAAGTTCAAAATTGTAAAAATTG100                          TAATTATAAAATCATTAAAGTTAATAATAATATAAATAAAAAAATTTTTA150                          TCGCATTTAAACAAGTTTCTTAATAATTTTGAATTTTAATTTAGAAAATA200                          TAAAAATCTTGTTGATTTTTATATAATTTTTTAACCTTTTTTTTTTTTTT250                          TTTTTTTTTAGGAATATGGTAAAATTTAGTCCATCTATCAAAAATATAGA300                          AAAGGAAAAATTATGAAGAAAAAAGCAAGAAAATTCTTAAGACTA345                               MetLysLysLysAlaArgLysPheLeuArgLeu                                              1510                                                                           ACTTCGCTTACACTAGCGCCTTTTTCGGTCTTCACCACTCTTATT390                               ThrSerLeuThrLeuAlaProPheSerValPheThrThrLeuIle                                  152025                                                                         TCAGCTGGTTGTTTGCAAAAAAATTCTTTGCTTTCAGAAGTAAAT435                               SerAlaGlyCysLeuGlnLysAsnSerLeuLeuSerGluValAsn                                  303540                                                                         TATTTAGCCCTAGGTGATTCACTAACAGCTGGATTTAATGAAGAA480                               TyrLeuAlaLeuGlyAspSerLeuThrAlaGlyPheAsnGluGlu                                  455055                                                                         ACATACCGTGATTTTCAAGGTACTTTAGATAAAGATGGTAATTTAAGC528                            ThrTyrArgAspPheGlnGlyThrLeuAspLysAspGlyAsnLeuSer                               606570                                                                         GGTCAATCTTATCCTGCTTATTTTGCTTATTATCTACAAAAACTTAAT576                            GlyGlnSerTyrProAlaTyrPheAlaTyrTyrLeuGlnLysLeuAsn                               758085                                                                         AAGAATTCACTTGTTTCTTATGATAATTTGGCAATTTCTGGGACAACA624                            LysAsnSerLeuValSerTyrAspAsnLeuAlaIleSerGlyThrThr                               9095100                                                                        ACAGAAAACTGACTTTACCTTCTTAATCCAACCAAATATCCAAATGGA672                            ThrGluAsnTrpLeuTyrLeuLeuAsnProThrLysTyrProAsnGly                               105110115120                                                                   AAAATGAGCGATAATCCGTTAGTTACAAACTATTCAGGAAATGAAAAA720                            LysMetSerAspAsnProLeuValThrAsnTyrSerGlyAsnGluLys                               125130135                                                                      TATAATGAAATAGGCTCTGTTTTTGGTGATTTTAATAAGGATTCCTAT768                            TyrAsnGluIleGlySerValPheGlyAspPheAsnLysAspSerTyr                               140145150                                                                      CCTGGTTTAGTCGAAAAAGTTAAAAAAGCAAACCTTTTGACAATGTCA816                            ProGlyLeuValGluLysValLysLysAlaAsnLeuLeuThrMetSer                               155160165                                                                      GTGGGAGCTAACGATCCTTTTTTAGCAATTTTTAATGAATTTAAAAAA864                            ValGlyAlaAsnAspProPheLeuAlaIlePheAsnGluPheLysLys                               170175180                                                                      TGAGCAAGTATAATAAAACCAAAATCAGAGGAAGCAAAAAAATTACTA912                            TrpAlaSerIleIleLysProLysSerGluGluAlaLysLysLeuLeu                               185190195200                                                                   GATCCAAATGAAAGAGCGAATTTCCTAGCAGAAAAAGGAATGCTTTTA960                            AspProAsnGluArgAlaAsnPheLeuAlaGluLysGlyMetLeuLeu                               205210215                                                                      AAGGCGGAAGTTAATAAAAAAATTGAGGAAATAAACACAAATCTTGAT1008                           LysAlaGluValAsnLysLysIleGluGluIleAsnThrAsnLeuAsp                               220225230                                                                      AATTTAATTAAAGAATTAAAGGCGCTTAATCCAAAATTAAGTATAAAT1056                           AsnLeuIleLysGluLeuLysAlaLeuAsnProLysLeuSerIleAsn                               235240245                                                                      TTAGTTGGATATAAATTGCCAAATTCCGGTTTTATTAAGATTTTAAAG1104                           LeuValGlyTyrLysLeuProAsnSerGlyPheIleLysIleLeuLys                               250255260                                                                      TATCTTTTATATACTTATGCAAAAATTGAAACGGACTTTATCAATGAA1152                           TyrLeuLeuTyrThrTyrAlaLysIleGluThrAspPheIleAsnGlu                               265270275280                                                                   ATTCCCGAAAAAATTAACAAAATTATTCGTGAAACCGCCATTAAAAAT1200                           IleProGluLysIleAsnLysIleIleArgGluThrAlaIleLysAsn                               285290295                                                                      AAGGTAAATTATATTGATGTCTATGATAAAAGTATTTGAAATGATTCT1248                           LysValAsnTyrIleAspValTyrAspLysSerIleTrpAsnAspSer                               300305310                                                                      GATAAAAATTTAATGGCGAAAAATTTTGACTTCCACCCTTCAATTCAA1296                           AspLysAsnLeuMetAlaLysAsnPheAspPheHisProSerIleGln                               315320325                                                                      GGTTATAAAAAAATTGCTCACCAACTTTTGTTAAAATTAACTCTTGAC1344                           GlyTyrLysLysIleAlaHisGlnLeuLeuLeuLysLeuThrLeuAsp                               330335340                                                                      CAAGAAGAAAAAGATGATTCTAATGCTGAAGAGCTAAAAAATACTACA1392                           GlnGluGluLysAspAspSerAsnAlaGluGluLeuLysAsnThrThr                               345350355360                                                                   AATTTCGATGATTTTGATGAGAATAAACCGACCTATTCCAAAGTTATT1440                           AsnPheAspAspPheAspGluAsnLysProThrTyrSerLysValIle                               365370375                                                                      GACCTAAGTGTTTTTGCAAAATCAAATAAAGAATTTCTTGAAAAATTA1488                           AspLeuSerValPheAlaLysSerAsnLysGluPheLeuGluLysLeu                               380385390                                                                      AACGAAAATAAGCAAACTAGTGAATTTATTGCTCAAAAATCCACTTTT1536                           AsnGluAsnLysGlnThrSerGluPheIleAlaGlnLysSerThrPhe                               395400405                                                                      GACACCGATCAAGAAGCTGCAATCAAAGACGACAAACGCACTTTT1581                              AspThrAspGlnGluAlaAlaIleLysAspAspLysArgThrPhe                                  410415420                                                                      GGAAATATAGTTCGAGAAATTGTATCTTTACCAATCTTCGATAAT1626                              GlyAsnIleValArgGluIleValSerLeuProIlePheAspAsn                                  425430435                                                                      TTTGATTTTAGAGAGTTAATACCTGTTAAAAATCCATTTGTAAAA1671                              PheAspPheArgGluLeuIleProValLysAsnProPheValLys                                  440445450                                                                      GCAATTATTAACAGCTATTTAGGGAAACCAGCTGGTTCTCTTATA1716                              AlaIleIleAsnSerTyrLeuGlyLysProAlaGlySerLeuIle                                  455460465                                                                      AAAGATATCGAACAACTCGAAAATAAAGTGAAAGATTACGCAAGA1761                              LysAspIleGluGlnLeuGluAsnLysValLysAspTyrAlaArg                                  470475480                                                                      CCTAATATCAAGATTTTCGATACAATTATTGACTCATTCATAAGA1806                              ProAsnIleLysIlePheAspThrIleIleAspSerPheIleArg                                  485490495                                                                      AAAATGGTAGCATTTTTTGCTGAATTAAACACTGATCAAGAAATA1851                              LysMetValAlaPhePheAlaGluLeuAsnThrAspGlnGluIle                                  500505510                                                                      AAAGAATTCAAAATGTCACCTCAAATACTATTTCTGACACTAAGA1896                              LysGluPheLysMetSerProGlnIleLeuPheLeuThrLeuArg                                  515520525                                                                      AATGCAATACTAAGTCCATTTGATTTAACTAAATTAAAAGACAGT1941                              AsnAlaIleLeuSerProPheAspLeuThrLysLeuLysAspSer                                  530535540                                                                      GCTACATTTAAAATTTTAATGAATCTCAAACCAGAACAAATATTA1986                              AlaThrPheLysIleLeuMetAsnLeuLysProGluGlnIleLeu                                  545550555                                                                      ACTCTACTAGGCCTAGGTAAAACCCCTTCAGTTCCTAAACCTGAA2031                              ThrLeuLeuGlyLeuGlyLysThrProSerValProLysProGlu                                  560565570                                                                      AAACCAAAAGATCAAGGTTCTATGCCACAAACAGATACTTCTAGT2076                              LysProLysAspGlnGlySerMetProGlnThrAspThrSerSer                                  575580585                                                                      CAAAAACAAGAAAGCGGAACAGGTTCAACAGATTCAACAAAAGCT2121                              GlnLysGlnGluSerGlyThrGlySerThrAspSerThrLysAla                                  590595600                                                                      ACAACTGAAAACCAAAAACCAGCTGAGCAAACAAATTCTTCTGAG2166                              ThrThrGluAsnGlnLysProAlaGluGlnThrAsnSerSerGlu                                  605610615                                                                      CAATCAAGTACCGATTCTAAATCAAACTAATTTTTTAATAACTTATA2213                            GlnSerSerThrAspSerLysSerAsn                                                    620625                                                                         ATTATAAAAAACCTAAACTTATTTCAGTTTAGGTTTTTATTTTCTAATTT2263                         CAAATTAGAAAATAAGACTTTCTAAAAAAGTCTTATTAAAATGTTAAAAA2313                         AACCTTGTTTTTTATAGACTTTTTTAAATTTTTTATTATAATATATAAGG2363                         AAAAATTTTAGTATTTCTGACTGTGAAATTATGAAGTTAATAAAAATTGA2413                         AATTGAAGGTTTTAAATCCTTTGCTGAACCTGTAAGTATTAAATTTGATG2463                         GTTCAATTGTTGGAATAATTGGGCCAAATGGCTCTGGAAAATCCAATATA2513                         AATGATGCAATTAAATGAGTTTTAGGCGAAAAATCAGTTAAACAATTACG2563                         GGGCCAAAATATGGATGATGTCATTTTTGCTGGCTCAAAAACAGTTATGC2613                         CTGTTAATAAAGCGATGGTAAAACTGACATTTTTAGATGAAACTCGTGAA2663                         GATAGTGCC2672                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 627 amino acid residues                                            (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (A) DESCRIPTION: predicted amino acid sequence of                              complete 627 residues of the P65                                               lipoprotein, derived from the                                                  nucleic acid sequence                                                          (iii) HYPOTHETICAL: no                                                         (iv) ANTI-SENSE: no                                                            (v) FRAGMENT TYPE: whole polypeptide                                           (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Mycoplasma hyopneumoniae                                         (B) STRAIN: J                                                                  (C) INDIVIDUAL ISOLATE:                                                        (D) DEVELOPMENTAL STAGE:                                                       (E) HAPLOTYPE:                                                                 (F) TISSUE TYPE:                                                               (G) CELL TYPE: unicellular bacterium                                           (H) CELL LINE:                                                                 (I) ORGANELLE:                                                                 (vii) IMMEDIATE SOURCE:                                                        (A) LIBRARY: Genomic in γ Charon 4A, γ GEM12                       (B) CLONE: γMhpJ25, γMhpJ35, γMhpJG35,                       pZJ25, pZJ25.1, pZJ25.14,                                                      pZJG35.1, pZJG35.12, pZJG35.13,                                                pZJG35.14                                                                      (viii) POSITION IN GENOME:                                                     (A) CHROMOSOME/SEGMENT: single chromosome                                      (B) MAP POSITION: unknown                                                      (C) UNITS: unknown                                                             (ix) FEATURE:                                                                  (A) NAME/KEY: 627 amino acid sequence                                          representing complete sequence                                                 (includingsignalsequence) of                                                   surface lipoprotein P65                                                        (B) LOCATION: entire derived coded sequence                                    (C) IDENTIFICATION METHOD: clone identified by immuno-                         detection of protein product                                                   with antiserum specific for P65;                                               residue sequence deduced from                                                  nucleic acid sequence                                                          (D) OTHER INFORMATION: immunogenic surface lipoprotein                         of no known function; C- terminus                                              exposed on external surface of                                                 cell; N-terminal signal sequence                                               (first29aminoacidresidues)                                                     cleaved during lipid modifi-                                                   cation process                                                                 (x) PUBLICATION INFORMATION:                                                   (A) AUTHORS: Mary F. Kim, Manijeh B. Heidari,                                  Susan J. Stull, Mark A.                                                        McIntosh, and Kim S. Wise                                                      (B) TITLE: Identification and Mapping of an                                    Immunogenic Region of Mycoplasma                                               hyopneumoniae p65 Surface                                                      Lipoprotein Expressed in                                                       Escherichia coli from a Cloned                                                 Genomic Fragment                                                               (C) JOURNAL: Infection and Immunity                                            (D) VOLUME: 58                                                                 (E) ISSUE: 8                                                                   (F) PAGES: 2637- 2643                                                          (G) DATE: August 1990                                                          (H) DOCUMENT NUMBER:                                                           (I) FILING DATE:                                                               (J) PUBLICATION DATE:                                                          (K) RELEVANT RESIDUES IN SEQ ID NO: From 1 to 627                              (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       MetLysLysLysAlaArgLysPheLeuArgLeuThrSerLeuThrLeu                               151015                                                                         AlaProPheSerValPheThrThrLeuIleSerAlaGlyCysLeuGln                               202530                                                                         LysAsnSerLeuLeuSerGluValAsnTyrLeuAlaLeuGlyAspSer                               354045                                                                         LeuThrAlaGlyPheAsnGluGluThrTyrArgAspPheGlnGlyThr                               505560                                                                         LeuAspLysAspGlyAsnLeuSerGlyGlnSerTyrProAlaTyrPhe                               65707580                                                                       AlaTyrTyrLeuGlnLysLeuAsnLysAsnSerLeuValSerTyrAsp                               859095                                                                         AsnLeuAlaIleSerGlyThrThrThrGluAsnTrpLeuTyrLeuLeu                               100105110                                                                      AsnProThrLysTyrProAsnGlyLysMetSerAspAsnProLeuVal                               115120125                                                                      ThrAsnTyrSerGlyAsnGluLysTyrAsnGluIleGlySerValPhe                               130135140                                                                      GlyAspPheAsnLysAspSerTyrProGlyLeuValGluLysValLys                               145150155160                                                                   LysAlaAsnLeuLeuThrMetSerValGlyAlaAsnAspProPheLeu                               165170175                                                                      AlaIlePheAsnGluPheLysLysTrpAlaSerIleIleLysProLys                               180185190                                                                      SerGluGluAlaLysLysLeuLeuAspProAsnGluArgAlaAsnPhe                               195200205                                                                      LeuAlaGluLysGlyMetLeuLeuLysAlaGluValAsnLysLysIle                               210215220                                                                      GluGluIleAsnThrAsnLeuAspAsnLeuIleLysGluLeuLysAla                               225230235240                                                                   LeuAsnProLysLeuSerIleAsnLeuValGlyTyrLysLeuProAsn                               245250255                                                                      SerGlyPheIleLysIleLeuLysTyrLeuLeuTyrThrTyrAlaLys                               260265270                                                                      IleGluThrAspPheIleAsnGluIleProGluLysIleAsnLysIle                               275280285                                                                      IleArgGluThrAlaIleLysAsnLysValAsnTyrIleAspValTyr                               290295300                                                                      AspLysSerIleTrpAsnAspSerAspLysAsnLeuMetAlaLysAsn                               305310315320                                                                   PheAspPheHisProSerIleGlnGlyTyrLysLysIleAlaHisGln                               325330335                                                                      LeuLeuLeuLysLeuThrLeuAspGlnGluGluLysAspAspSerAsn                               340345350                                                                      AlaGluGluLeuLysAsnThrThrAsnPheAspAspPheAspGluAsn                               355360365                                                                      LysProThrTyrSerLysValIleAspLeuSerValPheAlaLysSer                               370375380                                                                      AsnLysGluPheLeuGluLysLeuAsnGluAsnLysGlnThrSerGlu                               385390395400                                                                   PheIleAlaGlnLysSerThrPheAspThrAspGlnGluAlaAlaIle                               405410415                                                                      LysAspAspLysArgThrPheGlyAsnIleValArgGluIleValSer                               420425430                                                                      LeuProIlePheAspAsnPheAspPheArgGluLeuIleProValLys                               435440445                                                                      AsnProPheValLysAlaIleIleAsnSerTyrLeuGlyLysProAla                               450455460                                                                      GlySerLeuIleLysAspIleGluGlnLeuGluAsnLysValLysAsp                               465470475480                                                                   TyrAlaArgProAsnIleLysIlePheAspThrIleIleAspSerPhe                               485490495                                                                      IleArgLysMetValAlaPhePheAlaGluLeuAsnThrAspGlnGlu                               500505510                                                                      IleLysGluPheLysMetSerProGlnIleLeuPheLeuThrLeuArg                               515520525                                                                      AsnAlaIleLeuSerProPheAspLeuThrLysLeuLysAspSerAla                               530535540                                                                      ThrPheLysIleLeuMetAsnLeuLysProGluGlnIleLeuThrLeu                               545550555560                                                                   LeuGlyLeuGlyLysThrProSerValProLysProGluLysProLys                               565570575                                                                      AspGlnGlySerMetProGlnThrAspThrSerSerGlnLysGlnGlu                               580585590                                                                      SerGlyThrGlySerThrAspSerThrLysAlaThrThrGluAsnGln                               595600605                                                                      LysProAlaGluGlnThrAsnSerSerGluGlnSerSerThrAspSer                               610615620                                                                      LysSerAsn                                                                      625                                                                            __________________________________________________________________________ 

What is claimed:
 1. A vaccine for protecting a susceptible swine against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae comprising an immunogenic fusion protein in a suitable physiologically acceptable carrier, said fusion protein having a first amino acid sequence with the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2), or immunogenic fragments thereof said first amino acid sequence being fused to a second amino acid sequence.
 2. A method for inducing an immune response in a susceptible swine against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae, comprising administering to the swine a composition comprising an isolated and purified protein having the amino acid sequence depicted in FIG. 2 (SEQ ID NO:2), immunogenic fragments or immunogenic fusion proteins thereof in an amount effective for producing an immune response against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae.
 3. A vaccine for protecting a susceptible swine against mycoplasmal pneumonia caused by Mycoplasma hyopneumoniae comprising an immunogenic fusion protein in a suitable physiologically acceptable carrier, said fusion protein having a first amino acid sequence encoded by the DNA in FIG. 1 (SEQ ID NO:1) said first amino acid sequence being fused to a second amino acid sequence. 